The goal of this research is to develop novel, more effective therapies for the treatment of tuberculosis, including multidrug-resistant tuberculosis and related atypical mycobacterial infections. The complex cell wall of these gram-positive actinomycetes is their most characteristic feature and its biosynthesis is the target of some of the most effective antimycobacterial agents. A better understanding of the biochemical transformations used in cell wall elaboration will allow us to design novel interventions through the use of the tools of rational drug design and combinatorial chemistry. This project has focussed on understanding the biosynthetic relationships of various cell wall constituents through the use of the classical techniques of protein purification and analysis as well as genetic manipulation of various mycobacterial species. Many of these studies have involved mycolic acids, complex alpha-branched, beta-hydroxy fatty acids that are unique to mycobacteria which are heavily modified by a variety of functional groups. Modification reactions of mycolic acids are essential to proper functioning of the cell wall as a permeability barrier and as a mediator of mycobacteria-host cell interactions. We have identified a family of nine highly-related enzymes which are responsible for introducing these modifications and characterized their function. Surprisingly all of these enzymes appear to utilize a common chemical mechanism involving initial cation formation following methyl group addition to an olefinic mycolate precursor. This common intermediate offers a uniquely vulnerable target for intervention since inhibiting it would affect nine enzymes simultaneously, making the emergence of drug resistance very unlikely. We have cloned, overexpressed, and purified the enzymes which catalyze these reactions, characterized their activity in vitro and begun characterizing the substrate, product, and acyl carrier involved in these reactions. These studies have led to a detailed picture of how mycolic acids are biosynthesized and the point at which the modification reactions take place, both previously unknown. An additional are of focus of the project has been to study the mechanism of action of a commonly used second line antitubercular agent that affects cell wall synthesis at the point of attachment of the mycolic acids to the cell wall polysaccharide scaffolding. In collaboration with scientists at Stanford University we have examined patterns of differential gene expression using hybridization of cDNA derived probes to DNA microarrays designed to incorporate the entire genome of 4000 open reading frames from M. tuberculosis strain H37Rv. Using this technique we have examined temporal expression of genes in response to ethambutol, a drug that inhibits branching of the arabinogalactan and elaboration of the mycolic acid layer essential for cell wall structure. We have identified genes that are both induced and repressed and are characterizing many of these currently. Ethambutol is a relatively simple chemical structure composed of a 1,2-disubstituted symmetrical diamine. This compound was derived by optimization of an initial lead molecule by chemists at Lederle through the synthesis of about 2,000 symmetrical analogs. Although clinically useful the efficacy for this drug remains low and its full potential has not been realized. In order to validate our general goal of utilizing genomics through microarrays to guide discovery and lead optimization we have developed a solid-supported synthesis of analogs of ethambutol that allow us to access unsymmetrical analogs. Using techniques of split and pool combinatorial chemical synthesis we have now synthesized nearly 50,000 discrete analogs of ethambutol in pools of 10-20 compounds and screened them against promoter fusions to induced genes identified through microarrays. In collaboration with Sequella, Inc., under the terms of NIAID CRADA AI-0100 we hope to implement full-scale production of a library of 1 million ethambutol analogs. Currently we have identified chemically novel structures with activity comparable to or better than that of the parent molecule. - tuberculosis, mycobacteria, drug development, cell wall, and mycolic acids.